Thymic development is a highly choreographed differentiation process involving distinct lymphoid-stromal interactions in defined cortical and medullary locations. Lymphopoiesis of the T lineage in the thymus proceeds in a directional manner such that early double negative (DN) progenitors enter the perimedullary cortex and then move outward to the subcapsule while, conversely, double positive (DP) thymocytes flow inward toward the medulla. Such movement requires adhesion mechanisms for traction as well as attractive or repulsive cueing signals. We have performed in vivo global gene expression analysis of thymocyte developmental subpopulations and determined that the plxnd1 gene is rapidly repressed by ~ 100 fold as development proceeds from DP to single positive (SP) thymocytes. This gene encodes PlexinD1, an ill-defined member of the plexin family of axonal guidance molecules that bind secreted and cell-bound semaphorins to control cell fate in the nervous system. The expression pattern of other plexins in the thymus is distinct from that of Plexin D1. Furthermore, candidate semaphorin ligands (3F, 3G, 4B, 4D, 4G, 6D, and 7A) are under strict spatial transcriptional control in the thymus. Based on precedent with axonal guidance in the nervous system, we hypothesize that plexin-semaphorin interaction will allow for positional localization of various thymocyte subpopulations at specific developmental states. The purpose of this proposal is to generate animals in addition to the plxnd1-/- mutant, which results in neonatal lethality thus preventing analysis of T lymphoid development. The traditional plxnd1-/- will be used to analyze the consequences of plxnd1 disruption using plxnd1-/- ES cells and RAG-2-/- blastocyst complementation, as well as plxnd1-/- fetal liver transplantation. Utilizing transgenic mice in which plxnd1 expression is driven by the CD2 promoter, the effect of plxnd1 over-expression on thymocyte development and migration will be determined. Development of plxnd1-/- thymocytes within plxnd1 wild type thymic stroma will be analyzed using conditional, thymus-specific plxnd1 knockout strategies. The mouse models resulting from the implementation of the overexpression and conditional knockout strategies will greatly aid our analysis of the role PlexinD1 plays in thymic development. Project Narrative PUBLIC HEALTH RELEVANCE: The thymus is an organ of the immune system in which all T lymphocytes that protect a person against infectious diseases are generated. T lymphocytes are an important arm of the immune system acting to recognize and attack cells in an individual's body that may be infected by viruses, bacteria or may be malignant. A detailed understanding of this thymic development is important for public health and may offer new approaches for vaccine design.